Phytochemistry 61 (2002) 687692

www.elsevier.com/locate/phytochem

Bioactive terpenoids from sunower leaves cv. Peredovick1

Francisco A. Macasa,*, Ascension Torresa, Jose L.G. Galindoa, Rosa M. Varelaa,
Jose A. Alvarezb, Jose M.G. Molinilloa
a

Grupo de Alelopata, Departamento de Qumica Organica, Facultad de Ciencias, Universidad de Cadiz, C/. Republica Saharaui s/n,
Apdo. 40, 11510-Puerto Real, Cadiz, Spain
b
Departamento de Qumica Fsica, Facultad de Ciencias, Universidad de Cadiz, C/. Republica Saharaui s/n,
Apdo. 40, 11510-Puerto Real, Cadiz, Spain
Received 22 May 2002; received in revised form 5 August 2002

Abstract
The CH2Cl2 extract of dried leaves of Helianthus annuus L. cv. Peredovick1 has yielded, in addition to the known sesquiterpene
lactones annuolide E and leptocarpin, and the sesquiterpenes heliannuols A, C, D, F, G, H, I, the new bisnorsesquiterpene,
annuionone E, and the new sesquiterpenes heliannuol L, helibisabonol A and helibisabonol B. Structural elucidation was based on
extensive spectral (one and two-dimensional NMR experiments) and theoretical studies. The sesquiterpenes heliannuol A and
helibisabonol A and the sesquiterpene lactone leptocarpin inhibited the growth of etiolated wheat coleoptiles.
# 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Helianthus annuus L.; Compositae; Cultivar; Sunower; Sesquiterpenes; Annuionone E; Heliannuol L; Helibisabonols A, B; Allelopathy;
Coleoptile; Bioassay

1. Introduction
Chemical studies of Helianthus annuus L. have shown
that this species is a rich source of terpenoids, particularly
sesquiterpenoids (Spring et al., 1981; Macas et al., 1994,
1996) with a wide spectrum of biological activities including potential allelopathy (Spring et al., 1991; Ghisalberti,
1997; Macas et al., 1999b,c, 2000a,b). We have continued
our systematic studies of the allelopathic activity of different varieties of Helianthus annuus L. by analyzing
H. annuus L. cv. Peredovick1 CH2Cl2 dry leaf extract.
Herein, we report the isolation and structural elucidation of four new terpenoids, the bisnorsesquiterpene
(1), the 7,11-heliannane (2) and two sesquiterpenes (3, 4)
(Fig. 1), in addition to the known sesquiterpene lactones
annuolide E and leptocarpin; the 7,10-heliannanes
heliannuols C, D, F, and I; and the 7,11-heliannanes
heliannuols A, G and H.

Part 16 in the series Allelopathic studies in cultivar species; for

part 15 see Macas et al., 2000b.
* Corresponding author. Tel.: +34-956-016370; fax: +34-956016193.
E-mail address: famacias@uca.es (F.A. Macas).

Bioactivity of the isolated compounds was tested

(excepting heliannuol L due to the low amount
obtained) using the etiolated wheat coleoptiles bioassay
(Hancock et al., 1964) in a range of 10
410
6 M.
Helibisabonol A (3) was also tested at 10
3 M.

2. Results and discussion

The CH2Cl2 dry leaf extract of Helianthus annuus L
cv. Peredovick1 was chromatographed on a silica gel
column (Flash chromatography) using hexane-acetone
mixtures of increasing polarity. Medium polar fractions
yielded 13 compounds: the sesquiterpene lactones
annuolide E (Macas et al., 1993a) and leptocarpin
(Rolando et al., 1979; Delgado et al., 1984); the bisnorsesquiterpene annuionone E (1); heliannuols A, C, D, F,
G, H, I (Macas et al., 1993b, 1994, 1999a) and L (2);
and the sesquiterpenes helibisabonol A (3) and helibisabonol B (4). The compounds 14 are rst reported in
the literature (Fig. 1). Spectroscopic data of the known
compounds were identical to those previously reported.
Annuionone E (1) was isolated as colourless oil. Its
IR spectrum showed the presence of a hydroxyl group

0031-9422/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00370-9

688

F.A. Macas et al. / Phytochemistry 61 (2002) 687692

Table 1
1
H NMR data of compounds 14a
H

1b

2
20
3
4
40
6
7
70
8
80
9
90
10
11
110
12
13
14
15

2.33 d
2.36 d
2.38
2.21
1.62
1.75
1.35
1.68
1.64
3.82

dd
dd
dd
m
m
m
m
ddq

1.23 d
3.63 d
3.56 dd
1.31d s
1.07d s

2b

3c

4c

6.77 s

6.51

6.52

6.56 s
3.49 dq

6.59
3.06 ddq

6.49
3.70 dq

4.32 ddd

5.81 dd

2.35 ddd
1.62 ddd
3.70 brdd

1.74
1.55
1.45
1.23
3.66

1.15
1.13
1.21 d
2.18 s

1.04d
1.03d
1.09
2.04

dddd
dddd
dddd
m
dd

5.46 dd
3.78 d

1.07d
1.02d
1.26
2.1

J (Hz) 1: 220 =17.0; 440 =17.7; 40 -6=1.2; 67=9.9; 670 =6.5; 9

8=6.1; 980 =6.0; 109=6.2; 11110 =7.9; 110 -4=2.9; 2: 78=3.5; 7
14=7.5; 89=6.0; 890 =9.0; 990 =14.3; 910=6.1; 90 -10=2.5; 3: 7
8=7.0; 780 =7.6; 714=5.1; 880 =12.4; 89=5.5; 890 =9.8; 80 9=8.7; 80 -90 =5.8; 990 =13.1; 109=1.4; 1090 =4.9; 4: 78=6.6; 7
14=6.6; 89=15.5; 910=7.7.

Fig. 1. Terpenoids from Helianthus annuus, cv. Peredovick1 used in

wheat coleoptile bioassay.

(3433 cm
1) and a carbonyl group (1680 cm
1). The

molecular ion at m/z 226.1598 in the HREIMS spectrum along with the 13C NMR data (Table 2) was in
good agreement with the molecular formula C13H22O3
(calculated mass 226.1569). The 1H NMR 2D COSY
spectrum showed two series of correlations: the rst one
connects signal at
, 2.38 (H-4, dd) with those at
2.21
(H-40 , dd) and
3.56 (H-110 , dd); H-110 is connected with
H-11(
3.63, d). In the second set of correlations H-9 (

3.82, ddq) was coupled with H-8 (
1.68; m), H-80 (

1.64, m), and H-10 (
1.23, d); H-8 and H-80 were coupled with H-7 (
1.75, m) and H-70 (
1.35, m), and these
with H-6 (
1.62, dd). These correlations, along with the
presence of three methyl groups at
1.23 (d, J10,9=6.2
Hz; H-10),
1.31 (s) and
1.07 (s) (H-12, H-13), and
chemical shifts assigned to H-2, H-20 , H-4, H-40 and H-9
allow us to propose an ionane skeleton with a carbonyl
group at C-3 (
209.6, s) and a hydroxyl group at C-9 (

67.9, d).
On the other hand, the lack of any signal for H-5 in
the 1H NMR spectrum (Table 1), the chemical shifts of
H-11 and H-110 signals, those for C-11 (
78.3) and C-5
(
83.5), and the molecular formula C13H22O3 suggested

a
399. 952 MHz, CDCl3 signal of residual CHCl3, centered at
7.25
and (CD3)2CO signal residual (CH3)2CO centered at
2.04. Multiplicities are not repeated if identical with those in the preceding column.
b
The spectrum was recorded in CDCl3 as solvent.
c
The spectrum was recorded in (CD3)2CO as solvent.
d
These values may be interchanged within the same column.

Table 2
13
C NMR data of compounds 1, 3 and 4a
C

1b

3c

4c

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

43.4 s
49.4*d t
209.6 s
48.6* t
83.5 s
53.6 d
21.4 t
38.5 t
67.9 d
20.7 q
78.3 t
24.8** q
23.7** q

132.1
147.5* s
117.9 d
121.9 s
148.8*
113.3
29.8 d
35.4 t
24.4 t
79.2 d
72.4 s
25.5*
29.0*
21.1 q
15.4 q

132.1
147.5*
117.9
121.9
148.8*
113.3
28.9
138.2 d
127.7 d
31.9
72.4
26.3*
26.5*
20.8
15.4

a
100.577 MHz, signals of CDCl3 and (CD3)2CO centered at
77.0
and
206.0 respectively. Degree of protonation was obtained by APT
heteronuclear multipulse programs; multiplicities are not repeated if
identical with those in the preceding column.
b
The spectrum was recorded in CDCl3 as solvent.
c
The spectrum was recorded in (CD3)2CO as solvent.
d
*, **: these values may be interchanged within the same column.

F.A. Macas et al. / Phytochemistry 61 (2002) 687692

the presence of an oxirane ring placed at C-5 and C-11,

consistent with related 5,11-oxirane bisnorsesquiterpenes (Macas et al., 1998). The relative stereochemistry at C-5 and C-6 was assigned as 5S*,6R*
based on the long-range COSY correlations observed
for H-4 and H-110 and for H-40 and H-6. These couplings must be explained by a 0 W0 path, as C-7 and C-11
apparently adopt a trans conguration.
PM3 semiempirical calculations (Stewart, 1989)
showed that all most stable conformers with such a relative stereochemistry had a diaxial orientation for protons
H-40 and H-6. Moreover, protons H-4 and H-110 should
adopt a planar disposition (theoretical  179.5 for H-40 ,
C-4, C-5, C-6 and-176.2 for H-6, C-6, C-5, C-4) which
are in good agreement with the observed coupling constants according with Karpluss rule (Table 3).
With regard to the chiral centre at C-9, NOE studies
did not allow to establish any relative conguration
because of the alkylic chain nature and its free rotation
status. An S* relative stereochemistry is proposed based
on the similarity of experimental coupling constants and
theoretical angles obtained using PM3 calculations for
those conformers with such stereochemistry (Table 3).
Heliannuol L (2) showed a HREIMS with a molecular ion at m/z 266.1525 consistent with a molecular
formula C15H22O4. Additional peaks at m/z 248
[MH2O]+ and the absorption band at 3400 cm
1 in the
IR spectrum, suggested the presence of associated
hydroxyl groups (Pretsch et al., 2000).
Typical signals of heliannuols (Macas et al., 1994),
were observed in the 1H NMR spectrum [H-3 (
6.77, s),
H-6 (
6.56, s), and H-15 (
2.18, s)] (Table 1). The structure was supported by the correlations found in the 1H
NMR 2D COSY spectrum: H-14 (
1.21, d) with H-7 (

3.49, dq); H-7 with H-8 (
4.32, ddd) geminal to a hydroxyl
group; H-8 with H-9 (
2.35, ddd) and H-90 (
1.62, ddd);
and, nally, H-9 and H-90 with H-10 (
3.70, brdd). The
multiplicity and chemical shifts of H-12 (
1.15, s) and H13 (
1.13, s) along with the lack of signals corresponding
to H-11 is in good agreement with a seven- or eight
membered heterocyclic ring heliannuol (Macas et al.,
1994). The relative stereochemistry at chiral centres C-7
and C-8 was established as 7S*,8R* based on the positive
NOE eect observed between H-7 and H-8 signals.
After a non conclusive series of NOEs experiments
conducted to establish the stereochemistry at C-10, a
conformational study of the four possible isomers
(seven- or eight membered ring and R or S conguration at C-10) was pursued (Fig. 2). The search of the
most stable conformation of each isomer was made
using GMMX (PCMODEL, 2000) and then, rening it
by PM3 semiempirical calculations. The dihedral angles
obtained for the most stable conformer of each structure compared with the experimental values of coupling
constants are presented in Table 3. A clear correspondence can be pointed out between theoretical angles and

689

Fig. 2. Dierent possible isomers of heliannuol L (2).

coupling constants of the stereostructure 2d (J (Hz):

7,8=1.6; 8,9=3.8; 8,9=9.6; 9,10=6.2; 9,10=1.2)
and the experimental values for 2. The lack of any conformational equilibrium for the eight member ring, previously observed for the heliannuol A (Macas et al.,
1993b) can be explained through the probable existence
of hydrogen bonds between the heterocyclic oxygen and
the corresponding hydroxyl groups at C-8 and C-10
(Fig. 3), consistent with the typical broad band absorption observed in the IR at 3400 cm
1.
Helibisabonol A (3), its 13C NMR spectrum (Table 2)
showed six signals assigned to an aromatic ring
132.1
(C-1),
147.5* (C-2),
117.9 (C-3),
121.9 (C-4),

148.8* (C-5), and
113.3 (C-6). Additional two signals

Table 3
Observed coupling constants vs  obtained for the most stable conformers of compounds 14
Protons Observed
J (Hz)
(1) Annuionona E

67
670
89
80 9
4110

 (Theoretical)
137.4
91.5
93.6
87.6
174.7

9.9
6.5
6.1
6.0
2.9
2a

(2) Heliannuol L

(3) Helibisabonol A

(4) Helibisabonol B

2b


2c


2d


78
89
89
910
910

3.5
6
9
6.1
2.5

73.7
66.5
61.6
80.5



60.4 162.2
51.2 112.3
53.5 46.4 165.2
3.2



75.7
44.8 166.9
44.9
37.9 160.2 55.0 69.1

78
780
910
90 10
78
910

7.0
7.6
1.4
4.9
6.6
7.7

70.7
173.7
73.8
40.5
52.8
31.2

690

F.A. Macas et al. / Phytochemistry 61 (2002) 687692

presence of such a double bond induced a downeld

shift of the signal corresponding to H-10 (
3.78) and
H-7 (
3.70) in comparison with those of 3 (H-10,
3.66
and H-7,
3.06).
The relative stereochemistry of the chiral centres C-7
and C-10 is proposed as 7R*,10R* based on the comparison between experimental coupling constants of
protons H-7 and H-10 with the theoretical values
obtained for the most stable conformers of each of the
possible isomers 7R*, 10R* and 7R*, 10S* (Table 3).
2.1. Bioassays

Fig. 3. NOE eects for heliannuol L (2), on the minimum energy

conformer obtained with theoretical PM3 calculations.

were in good agreement with aliphatic carbons attached

to oxygen,
79.2 (C-10) and
72.4 (C-11).
The HREIMS of 3 showed a molecular ion at m/z
268.1671 in agreement with the molecular formula
C15H24O4 (calculated mass, 268.1675). An additional
peak at m/z 251.1626 [MOH]+ and the absorption
band in the IR spectrum at 3364 cm
1 (OH), suggested
the presence of a hydroxyl group.
The 1H NMR recorded in CD3OD (Table 1), showed
signals at
6.59,
6.51 (1H each, s, H-6 and H-3), corresponding to an 1,2,4,5-tetrasubstituted benzene ring,
an aromatic methyl group
2.04 (3H, s, H-15) and two
methyl groups located at an oxygenated quaternary
carbon (
1.04, 3H, s and
1.03, 3H, s; H-12 and H-13).
Structure 3 was further supported by correlations
found in the 1H NMR 2D COSY spectrum, which
showed the following series of connectivities in the
alkane side chain: the signal of H-10 (
3.66, dd) was
coupled with H-9 (
1.45; dddd) and with H-90 (
1.23,
m); H-9 and H-90 were coupled with H-8 (
1.74; dddd)
and H-80 (
1.55, dddd); both H-8 and H-80 were coupled
with the benzylic proton H-7 (
3.06; ddq), and the latter
with the methyl group H-14 (
1,09; 3H, d).
In order to propose the stereochemistry at the chiral
centres C-7 and C-10, a conformational study using
semiempirical calculations (PM3) of the two possible
diasteroisomers of this compound (7R*,10S* and
7R*,10R*) was achieved. The comparison between the
theoretical dihedral angles in each most stable conformation and the experimental coupling constants
(Table 3) let us to propose the relative stereochemistry
7R*,10S* for the compound 3.
Helibisabonol B (4), the most signicant dierences
with respect to 3 were found in the 1H NMR (Table 1),
which showed two signals assigned to a vinylic system
with a trans disposition (
5.81, dd, J7,8=6.6, J8,9=15.5
Hz; H-8;
5.46, dd, J9,10=7.7, J9,8=15.5 Hz, H-9). The

The activity of heliannuols A, C, D (Macas et al.,

1994), and G (Macas et al., 1999a) and the sesquiterpene lactones annuolide E (Macas et al., 1993a) and
leptocarpin (Macas et al., 1999d) have been previously
evaluated in a standard phytotoxic bioassay using several Standard Target Species (STS) (Macas, et al.,
2000a,b).
In order to draw a complete picture of the isolated
compounds, they were tested (excepting 2 due to the low
amount obtained) using the etiolated wheat coleoptiles
bioassay (Hancock, et al., 1964) in a range of 10
410
6
M. Helibisabonol A (3) was also tested at 10
3 M.
This is a fast bioassay (24 h) and sensitive to a wide
range of bioactive substances including plant growth
regulators, herbicides (Cutler, 1984), antimicrobials,
mycotoxins, and assorted pharmaceuticals (Jacyno and
Cutler, 1993).
The growth of etiolated wheat coleoptiles was signicantly inhibited (P < 0.01) 33, 23 and 18% respectively with 10
4 M solutions of heliannuol A (6),
heliannuol D (7) and leptocarpin (5), while helibisabonol A (3) inhibited 27% at 10
3 M, all relative to control (11  0.7 mm). The observed activity for leptocarpin
(5) can be related with the exibility of this molecule. It
has been reported that the dierent spatial arrangements that the molecule can adopt play an important
role in activity (Velasco, 2000), as well as the presence of
two electrophilic functions: the a-methylene-g-lactone
moiety and the oxirane ring (Spring and Hager, 1982;
Macas et al., 1992).

3. Experimental
3.1. General
1

H NMR and 13C NMR spectra were recorded at

399.952 and 100.577 MHz, respectively, on a Varian
UNITY-400 spectrometer and with CDCl3 and
(CD3)2CO as solvent. The resonances of residual CHCl3
and C3H6O at
H 7.25 and 2.04 and signals of CDCl3
and (CD3)2CO
c 77.0 and 206.0, respectively were used
as internal reference for 1H and 13C spectra. Mass

F.A. Macas et al. / Phytochemistry 61 (2002) 687692

spectra were obtained using a VG 1250 or a Kratos MS80 RFA instrument at 70 eV. The IR spectra were
recorded on a Bio-Rad FTS-7. Optical rotations were
determined using a Perkin-Elmer polarimeter model 241
set on the sodium D line. Column chromatography was
performed on Silica gel (3575 mesh), and TLC analyses
were carried out using aluminium precoated Silica gel
plates. For HPLC, LiChrosorb silica 60 was used in the
normal-phase mode with dierential refractometer (RI)
and UV detectors in a Hitachi L-6020A HPLC instrument. All solvents were spectral grade or distilled from
glass prior to use.
3.2. Plant material
Leaves of H. annuus L. cv. Peredovick1 were collected during the third plant development stage (Macas
et al., 1999d) (plants 1.2 m tall with owers, 1 month
before harvest) and were provided by Rancho de la
Merced, Agricultural Research Station (CIFA), Junta
de Andaluca, Jerez, Spain.
3.3. Extraction and isolation
Dry leaves (1.7 kg) were soaked in CH2Cl2 (fresh
plant: solvent, 1:3) for 24 h at 25  C in the dark. The
CH2Cl2 extract was fractioned by dry ash chromatography on silica gel using n-hexane:acetone mixts of
increasing polarity, yielding 12350 ml frs which were
reduced to 7 frs. (AG) after comparison by TLC.
Bioassays of the dierent fractions (Macas et al.,
1999d) with the dicots Lactuca sativa and Lepidium
sativum, and the monocots Allium cepa and Hordeum
vulgare allowed selecting fr. D for further study. Fr. D
was chromatographed using silica gel CC and eluted
with n-hexane-acetone mixts of increasing polarity.
After separation using HPLC with a LiChrosorb silica
60 column compounds annuolide E (2.1 mg), leptocarpin (4.5 mg), annuoinone E (1) (5.6 mg), heliannuol
A (3.3 mg), heliannuol C (2.0 mg), heliannuol D (2.3
mg), heliannuol F (1.8 mg), heliannuol G (1.6 mg),
heliannuol H (1.7 mg), heliannuol I (3.4 mg), heliannuol
L (2) (1.0 mg), helibisabonol A (3) (3.2 mg) and helibisabonol B (4) (3.1 mg) were obtained.
3.3.1. Annuionone E (1)
Colourless oil; [a]25
D +4.2 (c 0.1, CH3OH); IR (neat,
KBr) max 3433 (OH), 1698 (CO), 1271 (COC)
cm
1; 1H NMR data, see Table 1; 13C NMR data, see
Table 2; EIMS m/z (rel. int.): 226 [M]+ (9), 169
[MC3H5O]+ (19), 97 [C6H9O]+ (100); HREIMS m/z
226.1598 (calc. for C13O3H22, 226.1569).
3.3.2. Heliannuol L (2)
Colourless oil; IR (neat KBr) max 3400 (OH), 1109
(COC) cm
1; 1H NMR data, see Table 1; EIMS m/z

691

(rel. int.): 266 [M]+ (25), 248 [MH2O]+ (4), 233

[MH2OCH3]+ (32); HREIMS m/z 266.1525 (calc. for
C15H22O4, 266.1518).
3.3.3. Helibisabonol A (3)
Colourless oil; [a]25
D
44.9 (c 0.1, CH3COCH3); IR
(neat, KBr) max 3364 (OH), 2926 (CH, Ar), 1656
(CC, Ar) cm
1; 1H NMR data, see Table 1; 13C NMR
data, see Table 2; EIMS m/z (rel. int.): 268 [M]+ (28),
250 [MH2O]+ (17), 151 [MC6H13O2]+ (60); HREIMS
m/z 268.1671 (calc. for C15H24O4 268.1675).
3.3.4. Helibisabonol B (4)
Colourless oil; [a]25
D
7.2 (c 0.1, CH3COCH3); IR
(neat, KBr) max 3354 (OH), 2969 (CH, Ar), 1654
(CC, Ar) cm
1; 1H NMR data, see Table 1; 13C NMR
data, see Table 2; EIMS m/z (rel. int.): 266 [M]+ (16),
248 [MH2O]+ (11), 151 [MC6H11O2]+ (40); HREIMS
m/z 266.1508 (calc. for C15H22O4, 266.1518).
3.4. Coleoptiles bioassay
Wheat seeds (Triticum aestivum L. cv. Duro) were
sown in 15 cm diameter Petri dishes moisted with water
and grown in the dark at 22  1  C for 3 days (Hancock
et al., 1964). The roots and caryopsis were removed
from the shoots. The latter were placed in a Van der
Weij guillotine and the apical 2 mm were cut o and
discarded. The next 4 mm of the coleoptiles were
removed and used for bioassay. All manipulations were
performed under a green safelight (Nitsch and Nitsch,
1956). Compounds were predisolved in DMSO and
diluted to the nal bioassay concentration with a maximum of 0.1% DMSO. Parallel controls were also run.
Crude extracts, fractions, or pure compounds to be
assayed for biological activity were added to test tubes.
The assay was made in duplicate. Phosphate-citrate
buer (2 ml) containing 2% sucrose (Nitsch and Nitsch,
1956) at pH 5.6 was added to each test tube. Following
the placement of ve coleoptiles in each test tube, the
tubes were rotated at 0.25 rpm in a roller tube apparatus
for 24 h at 22  C in the dark. The coleoptiles were
measured by digitalization of their images. Data were
statistically analysed using the Welchs test (Martn
Andres and Luna del Castillo, 1990). Data are presented
as percentage dierences from control. Thus, zero
represents the control; positive values represent stimulation of the studied parameter, and negative values
represent inhibition.

Acknowledgements
This research was supported by the Direccion General
de Investigacion Cientca y Tecnica, Spain (DGICYT
PB980595).

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F.A. Macas et al. / Phytochemistry 61 (2002) 687692

References
Cutler, H.G., 1984. Fresh look at the wheat coleoptile bioassay. In:
Proceedings of the 11th Annual Meeting of the Plant Growth Regulator Society of America. PGRSA, Boston, MS, USA, pp. 19.
Delgado, G., Alvarez, L., Romo de Vivar, A., 1984. Terpenoids and a
avan-3-ol from Viguiera quinqueradiata. Phytochemistry 23, 675
678.
Ghisalberti, E.L., 1997. Biologically active cyanogenetic iridoid and lignan glycosides from Eremophila maculate. Fitoterapia 68, 303325.
Hancock, C.R., Barlow, H.W., Lacey, H.J., 1964. The east malling
coleoptile straight growth test method. J. Exp. Bot. 15, 166176.
Jacyno, J.M., Cutler, H.G., 1993. Detection of herbicidal properties:
scope and limitations of the etiolated wheat coleoptile bioassay.
PGRSA Quarterly 21, 1524.
Macas, F.A., Galindo, J.C., Massanet, G.M., 1992. Potential allelopathic activity of several sesquiterpene lactone models. Phytochemistry
31, 19691997.
Macas, F.A., Varela, R.M., Torres, A., Molinillo, J.M.G., 1993a.
Potential allelopathic guaianolides from cultivar sunower leaves,
var. SH-222. Phytochemistry 34, 669674.
Macas, F.A., Varela, R.M., Torres, A., Molinillo, J.M.G., 1993b.
Novel sesquiterpene from bioactive fractions of cultivar sunowers.
Tetrahedron Lett. 34, 19992002.
Macas, F.A., Molinillo, J.M.G., Varela, R.M., Torres, A., 1994.
Structural elucidation and chemistry of a novel family of bioactive
sesquiterpenes: heliannuols. J. Org. Chem. 59, 82618266.
Macas, F.A., Torres, A., Molinillo, J.M.G., Varela, R.M., Castellano,
D., 1996. Potential allelopathic sesquiterpene lactones from sunower leaves. Phytochemistry 43, 12051215.
Macas, F.A., Varela, R.M., Torres, A., Oliva, R.M., Molinillo,
J.M.G., 1998. Bioactive norsesquiterpenes from Helianthus annuus
with potential allelopathic activity. Phytochemistry 48, 631636.
Macas, F.A., Varela, R.M., Torres, A., Molinillo, J.M.G., 1999a.
New bioactive plant heliannuols from cultivar sunower leaves.
J. Nat. Prod. 62, 16361639.
Macas, F.A., Varela, R.M., Torres, A., Molinillo, J.M.G., 1999b.
Potentiality of cultivar sunowers (Helianthus annuus L.) as source of
natural herbicide models. In: Inderjit Dakshini, K.M.M., Foy, Ch.L.
(Eds.), Principles and Practices in Plant Ecology. Allelochemical
Interactions. CRC Press LLC, Boca Raton, FL, USA, pp. 531550.
Macas, F.A., Molinillo, J.M.G., Varela, R.M., Torres, A., Galindo,
J.C.G., 1999c. Bioactive compounds from the genus Helianthus. In:

Macas, F.A., Galindo, J.C.G., Molinillo, J.M.G., Cutler, H.G.

(Eds.), Recent Advances in Allelopathy, Vol. I. A Science for the
Future. Cadiz University Press, Cadiz, Spain, pp. 121134.
Macas, F.A., Oliva, R.M., Varela, R.M., Torres, A., 1999d. Allelochemicals from sunowers leaves cv. Peredovick. Phytochemistry
52, 613621.
Macas, F.A., Castellano, D., Molinillo, J.M.G., 2000a. In the search
for a standard bioassay for allelopathic studies of phytotoxicity.
Selection of standard target species (STS). J. Agr. Food Chem. 48,
25122521.
Macas, F.A., Varela, R.M., Torres, A., Molinillo, J.M.G., 2000b.
Potential allelopathic activities of natural plants heliannanes: a
proposal of absolute conguration and nomenclature. J. Chem.
Ecol. 26, 1732186.
Martn Andres, A., Luna del Castillo, J. de D., 1990. Bioestadstica
para las Ciencias de la Salud, 3rd ed. Norma, Madrid.
Nitsch, J.P., Nitsch, C., 1956. Studies on the growth of coleoptile and
rst internode sections. A new sensitive, straight-growth test for
auxins. Plant Physiol. 31, 94111.
PCMODEL 7.5., 2000. Serena Software, Bloomington, Indiana.
Pretsch, E., Clerc, T., Seibl, J., Simon, W., 2000. Tablas para la
Determinacion Estructural por Metodos Espectroscopicos.
Springer-Verlag, Iberica, Barcelona, Spain, p. 256.
Rolando Martnez, J., Ayamante, B.I.S., Nunez-Alarcon, J.A., Romo
de Vivar, A., 1979. Leptocarpin and 17,18-dihydroleptocarpin, two
new heliangolides from Leptocarpha rivularis. Phytochemistry 18,
15271528.
Spring, O., Albert, K., Gradmann, W., 1981. Annuithrin, a new biologically active germacranolide from Helianthus annuus. Phytochemistry 20, 18831885.
Spring, O., Hager, A., 1982. Inhibition of elongation growth by two
sesquiterpene lactones isolated from Helianthus annuus L. Possible
molecular mechanism. Planta 156, 433440.
Spring, O., Benz, A., Faust, V., 1991. Impact of downy mildew (Plasmopara halstedii) infection on the development and metabolism of
sunower. J. Plant Dis. Protect. 98, 597604.
Stewart, J.J.P., 1989. Molecular orbital calculations were carried out
using PM3 Hamiltonian as implemented in MOPAC 6.0. Geometries were fully optimized using the PRECISE keyword. J. Comput. Chem. 10, 209221.
Velasco, R. F., 2000. Transformaciones Qumicas en Germacranolidas. Modelos de Herbicidas Naturales. MSc Thesis, Universidad
de Cadiz, Spain.